Embryo-Toxicity and Teratogenicity of Derris elliptica Leaf Extract on Zebra Fish (Danio rerio) Embryos

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1 Available online at Tolentino and Undan Int. J. Pure App. Biosci. 4 (3): (2016) ISSN: DOI: ISSN: Int. J. Pure App. Biosci. 4 (3): (2016) Research Article Embryo-Toxicity and Teratogenicity of Derris elliptica Leaf Extract on Zebra Fish (Danio rerio) Embryos Josephine Joy V. Tolentino 1 * and Jerwin R. Undan 2 1 Graduate Student, Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120 Philippines 2 Molecular Biology and Biotechnology Laboratory, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120 Philippines *Corresponding Author jjvtolentino0126@gmail.com Received: Revised: Accepted: ABSTRACT The toxic effects of Derris eliptica or Opay were assessed against zebrafish embryos. Leaf extract of D. elliptica via hot water extraction and the zebra fish (Danio rerio) as the model organism were utilized for the assessment of its teratogenicity and embryo-toxicity potential. Two concentrations of 0.05% (T 2 ) and 0.50% (T 3 ) of leaf extract were used, while embryo water medium was used as the control (T 1 ). Percent mortality was observed after 12, 24, and 48 hours. In addition, percent hatchability was observed after 48 hours only. The number of heart beats per minute of the zebra fish embryo within 48 hours post treatment was also recorded. The results showed that zebra fish embryos treated with 0.05% extract showed reduced hatchability rate, lower heartbeat rate and delayed formation compared with the control. The adverse effect of 0.50% treatment was still severe resulting in undeveloped head and tail region, coagulation and death of embryos. It was concluded that teratogenic and lethal effects on zebra fish increased with the increase in the dose concentration of leaf extracts of D. elliptica. Further investigation on this plant species affecting other animals with ethanol and acetone extracts are also recommended. Key words: Teratogenic, Embryo, Zebra fish, Derris elliptica, Hot water leaf extract INTRODUCTION Some plants are known to possess piscicidal activities or fish poisons. Such plants are used by some communities for fishing. In an indigenous community of Ikalahan tribe, Sta. Fe, Nueva Vizcaya, Philippines, a known wild type of plant, Derris elliptica or Opay in their dialect, is commonly used as a fish poison, since it has been known that the plant leaves had toxic effects to fishes. D. elliptica belongs to the family Fabaceae characterized as a large climber which is cultivated mainly for its roots as a source of insecticide (Rotenone) in some tropical countries 1. Some of the potential uses of piscicides are for cultural, commercial and environmental reasons; though most countries prohibit the use of piscicides in large scale killing of fish, but for some countries like Africa, South America, Philippines and areas in South Pacific, fish poisons are used to catch fish for food 2. Cite this article: Tolentino, J.J.V. and Undan, J.R., Embryo-Toxicity and Teratogenicity of Derris elliptica Leaf Extract on Zebra Fish (Danio rerio) Embryos, Int. J. Pure App. Biosci. 4(3): (2016). doi: Copyright June, 2016; IJPAB 16

2 The present study intends to assess the gross morphological endpoints of zebrafish and determine any abnormalities as affected by different concentrations of D. elliptica leaf extract. Establishment of this plant s leaf extract concentration that can possess teratogenic and lethal effects on zebrafish is also aimed in this study. was doled out into a 12-well ELISA plate using a micropipette. Three fertilized embryos were placed into each well per replicate using a sterile plastic dropper. Plates were incubated at 26 ± 1 C. Microscopic Observation and Analyses The fertilized embryos were observed first under a light microscope before placing in treatments. For each of the treatments, the teratogenic activity of the D. elliptica plant extract was examined through the aid of a compound microscope after 12, 24, and 48 hours after the incubation period. Evaluation of toxicological and morphological endpoint of zebrafishes was based on parameters established by Nagel (1994) as: teratogenic (malformation of head, malformation of tail, growth retardation, scoliosis/flexure, stunted tail, and limited movement); lethal (coagulation, tail not detached, no somites and no heartbeat); and normal. Presentation of Data and evaluation of mortality and hatchability rates of zebrafish The data recorded were used to (a) assess gross morphological endpoints of zebrafishes; (b) determine Percent mortality (characterized by the number of coagulated zebrafish embryos after 12, 24, and 48 hours post-treatment application (hpta)) computed using the formula: MATERIALS AND METHODS Preparation of Plant Leaf Extract The collected leaves of D. elliptica from Imugan, Sta. Fe, Nueva Vizcaya were sun-dried, shredded into smaller pieces and were further finely crushed until powdery using sterile blender. 20g of powdered leaves was placed in a sterile flask with 200 ml of distilled water and was evenly stirred. The solution was then subjected into a hot water bath maintained at 80 C for 2 hours and filtered. Spawning of Mature Zebrafish A glass aquarium filled with untreated, clean tap water with continuous supply of oxygen was provided to house adult male and female zebra fishes in 1:2 ratio based after a protocol 3. To instigate spawning of zebra fishes, the glass aquarium was covered with a black plastic sheet (dark condition) for 12 hours. Once spawning was finished, the eggs were subjected to a fluorescent bulb (lighted condition) for 12 hours as well. Fertilization of eggs takes place within. % = minutes after the introduction of light (Nagel,. 1998). The fertilized eggs were siphoned out of (c) Percent hatchability (characterized by the aquarium after 12 hours of fertilization using hatched zebrafish embryo after 48 hpta) a hose. The embryos were then rinsed three computed using the formula: times and placed in a clean watch glass containing embryo medium for microscopic. h h observations. % h = 100. Establishing Teratogenic assay D. elliptica hot water extract was diluted using (d) Number of heart beats per minute of embryo water. Two concentrations were zebrafish embryo in different treatment prepared separately as 0.05% (T 2 ) and 0.5% concentrations within 48 hpta was counted and (T 3 ). Pure embryo water was used to serve as averaged. control set-up and was labeled as 0% (T 1 ). T 2 Statistical Analyses has 500 µl of leaf extract mixed with ml Three replicates of each treatment were laid out of embryo water; while T 3 has 50 µl of leaf in a Complete Randomized Design (CRD) and extract in 9.95 ml of embryo water, were analyzed in One-Way Analysis of Variance respectively. (ANOVA). Significant treatment comparison at Teratogenicity and Embryo toxicity of D. 5% level of significance was determined using elliptica Extract Duncan Multiple Range Test (DMRT), LSD and After the complete examination of normal Tukey s Test using the SPSS version 21.0 embryos, 4 ml of each treatment concentration program. Copyright June, 2016; IJPAB 17

3 Tolentino and Undan Int. J. Pure App. Biosci. 4 (3): (2016) RESULTS Among the treatment groups, T1 (control) has normal development of embryos. At 48 hpta, hatching was completed for the two treatment concentrations (T1 and T2). The percent hatchability of embryos treated with T2 (0.05%) and T3 (0.5%)were significantly lower than that of the control after 48 hpta (Fig.1). There were no recorded hatchlings for 0.5% (T3) because ISSN: after 48 hpta, all embryos under this treatment group died. The highest number of heartbeat per minute was recorded in embryos incubated with embryo water (control) with a mean of 169 beats per minute (bpm). This was not significantly different from treatment 2 but with lower heartbeat rates of162 bpm. In contrast, no heartbeats were recorded on the treatment 3 due to death of embryos (Fig.2). Fig.3. Lethal and teratogenic effects of D. elliptica on zebrafish embryos. (A-B) Unformed head and tail regions were observed in embryos exposed to T3 (0.5%) after 24 hpta. (D) Coagulation and no visual heartbeat leading to subsequent death of the embryo was observed after 48 hpta in T3 (0.5%). Ufh- unformed tail; Uft- unformed head; and Co- coagulated. Different toxicological and morphological abnormalities in zebrafish embryos caused by D. elliptica leaf extract were noted as unformed head, unformed tail, coagulation and death for those exposed to 0.5% treatment concentrations of the extract (Fig.3 A-C). On the other hand, embryos exposed to T2 (0.05%) showed growth retardation and limited movements as to compare with the control group. Mortality rates of zebrafish embryos after subsequent exposure to Copyright June, 2016; IJPAB different extract concentrations led to early death of embryos specifically in T3 (0.5%). The mortality rates of embryos treated with T3 (0.5%) are significantly higher than T2 (0.05%) and control treatment groups respectively (Fig.4). No visual heartbeat and coagulation of the embryos were observed after 12 and 48 hpta of T3 (0.5%) of the leaf extract. All embryos of zebrafish were dead after 48 hours of post-treatment exposure of T3 (0.5%) of D. elliptica leaf extract. 18

4 Fig.4. Percent mortality of embryos after 12, 24 and 48 hours of treatment exposure to D. elliptica. Embryos treated with 0.5% extract concentration showed death of all embryos after 48hpta; which is significantly higher than the other two treatment groups. * means significant difference. DISCUSSION This study was able to test the embryo-toxic and teratogenic effects of the D. elliptica leaf extract against zebrafish as a model organism which could also be toxic for the development and growth of other vertebrate embryos which could be in a resemblance of effects among zebrafish embryos since the development of embryo in zebrafish is very similar with higher vertebrates 4. Teratology is the aspect of embryology involved with the study of abnormal development and a teratogen is any agent which overtly causes the production of a congenital defect or increases incidence of a particular congenital defect 5. D. elliptica, being a teratogen, was assessed for the extent of its teratogenic and lethal abilities wherein varying concentrations of the leaf extract affected the hatchability and even the rate of mortality on the zebrafish embryos. Apart from leaf extract, the aqueous root extract of D. elliptica was also found to be toxic to golden snail (Pomacea spp.) at 2000 ppm while the stem gave only 30% mortality at 10,000 ppm at the rate of 90kg/ha; and hence can be used as an effective snail control 6. Zebrafish embryos that were exposed to lower concentration (0.05%) of D. elliptica leaf extract exhibited growth retardation due to limited number of hatchlings produced after 48 hpta in contrast with the hatchability rate of embryos observed under normal embryo water medium (control group). The increased concentration to 0.05% severely affected the hatchlings and caused early death of embryos after 48 hpta. Thus, an inverse relationship between percent hatchability and concentration of the leaf extract could be perceived; where the increase in amount of concentration of the extract causes decrease in hatchability rate of the zebrafish embryos. Using one-way analysis of variance, there is statistical significant difference on the percent hatchability among the treatment samples. Low hatchability rate could be attributed to the delayed development of zebrafish embryos, and can therefore be one of the important aspects of the sub-lethal properties of the plant extract 4. Embryos exposed to T 3 (0.5%) extract concentration of the D. elliptica plant showed malformation (underdevelopment) of the head and tail regions, growth retardation, limited movement and coagulation after 24 hours of post-treatment application. The failure of organogenesis leads to the underdevelopment of the head and tail morphologies; which can be ascribed to the inhibition or disturbance of vital compounds essential for growth and development of embryos 4. The zebrafish embryos exposed to teratogen plant extract, which in this study was D. eliptica caused developmental delay, growth retardation and even early death of the embryos. In this present study, the embryos treated with 0.5% concentrations of the extract were characterized with delayed growth and Copyright June, 2016; IJPAB 19

5 development prior to malformations and early study and reviewers of this article for their death of the embryos which was statistically helpful comments and suggestions. significant using one-way analysis of variance. Further, the morphological endpoints related to REFERENCES the delayed development of zebrafish embryos 1. Starr, F., Starr, K. and Loope, L. Derris can be caused by the presence of boric acid (for elliptica. United States Geological Survey teratogenic concentrations) and valproic acid as Biological Resources Division Halekeala well as methoxyacetic acid (for non-teratogenic Field Station, Maui, Hawaii. (2003) 1-4. concentrations 7. No heartbeat rate was recorded 2. Burton, R., Cannon, J., Owen, N., and in T 3 (0.5%) concentration among all its Wood, S. Jour. of Chem. Edu. 81(10): replicates due to the early death of the embryos (2004). This study was able to reveal that in just 3. Nagel, R., In W. Heger, S. Jung, S. Martin. very minimal concentrations of D. elliptica such UBA Texteband Berlin: Umweltbundesamt. as 0.5% can be very toxic to fish embryos. Even (1998). at low concentrations, congenital deformities 4. Dulay, R., Kalaw, S., Reyes, R., and and malformations were observed and prolonged Cabrera, E. Ann. of Biol. Res.5 (6): 9- exposure to the leaf extract of this plant can be 14(2014). fatal to the embryos. Further investigation on 5. Moore, K.S The Developing Human. this plant species affecting other vertebrates 9 th edition. Saunders Elsevier, Inc. using ethanol and acetone extracts are also Canada suggested for future studies. 6. Maini, P. and Morallo-Rejesus,R. Phil. Jour. of Sci.64: (1993). ACKNOWLEDGEMENT 7. Teixido, E., Pique, E.; Gomez-Catalan, J., The authors thank DOST ASTHRDP SEI for Llobet, JM. Toxicol in Vitro, 27: providing financial assistance on this research (2013). Copyright June, 2016; IJPAB 20